Characterizing the potential of the non-conventional yeast Saccharomycodes ludwigii UTAD17 in winemaking

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microorganisms

Article

Characterizing the Potential of the Non-Conventional

Yeast Saccharomycodes ludwigii UTAD17

in Winemaking

Marcos Esteves1,2, Catarina Barbosa1,2,3 , Isabel Vasconcelos4, Maria João Tavares5 , Arlete Mendes-Faia1,2, Nuno Pereira Mira5and Ana Mendes-Ferreira1,2,*

1 _{WM&B—Laboratory of Wine Microbiology & Biotechnology, Department of Biology and Environment,}

Universidade de Trás-os-Montes e Alto Douro, 5000-801 Vila Real, Portugal; mjcesteves@utad.pt (M.E.); crbarbosa@utad.pt (C.B.); afaia@utad.pt (A.M.-F.)

2 _{BioISI—Biosystems & Integrative Sciences Institute, Campo Grande, 1749-016 Lisboa, Portugal}

3 _{CoLAB Vines&Wines - National Collaborative Laboratory for the Portuguese Wine Sector, Associação para o}

Desenvolvimento da Viticultura Duriense (ADVID), Edifício Centro de Excelência da Vinha e do Vinho, Régia Douro Park, 5000-033 Vila Real, Portugal

4 _{CBQF/Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Universidade Católica}

Portuguesa, 4169-005 Porto, Portugal; ivasconcelos@porto.ucp.pt

5 _{iBB - Institute of Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico,}

Universidade de Lisboa, 1049-001 Lisboa, Portugal; tavares.mja@gmail.com (M.J.T.); nuno.mira@tecnico.ulisboa.pt (N.P.M.)

* Correspondence: anamf@utad.pt; Tel.:+351-259-350-975

Received: 30 September 2019; Accepted: 18 October 2019; Published: 23 October 2019  Abstract:Non-Saccharomyces yeasts have received increased attention by researchers and winemakers, due to their particular contributions to the characteristics of wine. In this group, Saccharomycodes ludwigii is one of the less studied species. In the present study, a native S. ludwigii strain, UTAD17 isolated from the Douro wine region was characterized for relevant oenological traits. The genome of UTAD17 was recently sequenced. Its potential use in winemaking was further evaluated by conducting grape-juice fermentations, either in single or in mixed-cultures, with Saccharomyces cerevisiae, following two inoculation strategies (simultaneous and sequential). In a pure culture, S. ludwigii UTAD17 was able to ferment all sugars in a reasonable time without impairing the wine quality, producing low levels of acetic acid and ethyl acetate. The overall effects of S. ludwigii UTAD17 in a mixed-culture fermentation were highly dependent on the inoculation strategy which dictated the dominance of each yeast strain. Wines whose fermentation was governed by S. ludwigii UTAD17 presented low levels of secondary aroma compounds and were chemically distinct from those fermented by S. cerevisiae. Based on these results, a future use of this non-Saccharomyces yeast either in monoculture fermentations or as a co-starter culture with S. cerevisiae for the production of wines with greater expression of the grape varietal character and with flavor diversity could be foreseen.

Keywords: Saccharomycodes ludwigii; non-Saccharomyces; wine fermentation; mixed-culture; wine aroma

1. Introduction

Inoculation with active dry yeasts of Saccharomyces cerevisiae is a common practice in most wine-producing regions since the middle of the 20th century, to assure prompt and reliable fermentations and wines with a consistent and predictable quality. The main drawback of this practice is the loss of the typical distinctive characteristics found in wines obtained by spontaneous fermentation,

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carried out by winery- and grape-resident yeasts [1–5]. Grape-must microbiota is dominated by non-Saccharomyces yeast strains which have for long been considered spoilage agents, not only due to their low fermentative ability but also because of the assumption that they overproduce off-flavor compounds, such as acetic acid, acetaldehyde, acetoin, or ethyl acetate [1,6]. However, the demonstration that these negative traits are species and strain-dependent [7] and that some non-Saccharomyces yeasts even exhibit beneficial traits, not found in S. cerevisiae [8–14] have led winemakers to take a fresh look at these formerly disregarded species. In this line, over the last years, a massive number of studies searching for autochthonous non-Saccharomyces strains that might impart a unique aroma complexity or mouthfeel to wines, while expressing terroir-associated characteristics have been published [15–23]. Among the most studied non-Saccharomyces yeasts are the members of the genus Hanseniaspora (H. uvarum [24–28], H. guillermondii [24–26,29,30], and H. vineae [25,31–34]), Metschnikowia pulcherrima [21,33,35–37], Torulaspora delbrueckii [38–43], Kluyveromyces/Lachancea thermotolerans [44–48], and Starmerella bacillaris (formerly Candida stellata/Candida zemplinina) [27,49–53]. In addition to their contribution to the enhancement and diversification of wine aroma, it was found that these yeasts might display other oenological relevant traits, such as increased glycerol, mannoprotein, and total acidity contents [16,46,52,54,55], contributing to color stability [56,57] and reducing volatile acidity or ethanol levels [21,33,36,38,58]. In this context, the potential of developing starter cultures based on non-Saccharomyces yeast species has flourished in the wine world and several non-Saccharomyces strains that can be used as starter cultures are now commercially available [10,59]. Nevertheless, there are still a number of species whose potential in winemaking remains to be discovered. One such species is Saccharomycodes ludwigii (S. ludwigii), a bipolar budding yeast, first isolated from deciduous trees in Europe [60], which has a long history as a spoilage agent in winemaking. This yeast is rarely found in grapes but appears to be a usual contaminant of sulfite-preserved musts [6]. It has also been found in wines, at the end of the alcoholic fermentation or during storage [61], where it contributes for sedimentation or cloudiness formation [62]. The persistence of S. ludwigii in wineries is largely explained by its high tolerance to sulfur dioxide [63] and ethanol [7]. Regardless of its association with spoilage, S. ludwigii has been proposed as a starter-culture for the production of feijoa fermented beverages [61], cider [64], and low-alcohol or non-alcoholic beers [65,66]. In winemaking, the few studies undertaken with this yeast have shown that it could also be promising, since, depending on the strain, S. ludwigii possesses a good fermentative capacity [7] and is able to shape the aroma profile [8,18] and mouthfeel perception of wines [52]. Thus, the aim of this work was to examine the oenological potential of S. ludwigii UTAD17, an indigenous Douro Wine Region strain whose genome sequence has been recently released [67]. For this purpose, besides phenotyping the strain for relevant oenological traits, fermentations of a natural grape-must were performed, either in pure or in co-culture with S. cerevisiae. The growth and fermentation behavior, as well as the analytical profiles of the final wines, were also evaluated, revealing that this strain could be useful for tailoring wines with enhanced varietal characters.

2. Materials and Methods

2.1. Yeast Strains and Maintenance Conditions

The yeast S. ludwigii UTAD17, an autochthonous Douro Wine Region strain isolated in our laboratory [67], and the S. cerevisiae Lalvin QA23 (Lallemand-Proenol 4410-308 Canelas, Portugal), obtained from the market as an active dried yeast, were used in this study. Yeasts were routinely maintained at 4◦C on Yeast Extract Peptone Dextrose agar plates (YPD) containing per liter: 20 g of glucose, 10 g of peptone, 5 g of yeast extract, and 20 g of agar from stocks stored at −80◦C. Prior to use, the yeasts were transferred to a new slant of YPD and incubated for 24–48 h at 28◦C, unless otherwise stated.

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Microorganisms 2019, 7, 478 3 of 16

2.2. Phenotypic Characterization

S. ludwigii UTAD17 was screened for relevant enological features [68]. The evaluation of stress resistance and the activity of the enzymes of enological interest was performed as described in [21]. Briefly, for all assays, after growth in YPD medium until the mid-exponential growth phase, the yeast strain was inoculated in the appropriate culture media for stress tolerance and enzymatic activities evaluation. YPD agar plates without a stress agent, was used as the control. Accordingly, the following concentrations were used—6%, 9%, or 12% (v:v−1) of ethanol; 1, 2, or 4 mM of sulfur dioxide (SO2); 0.5, 1, or 2 mM of copper, supplied as copper sulfate; and 0.25, 0.5, or 1 mM of H2O2. Yeast growth in the presence of cerulenin or 5,50,500-trifluoro-d,l-leucine (TFL) was screened to evaluate the potential to produce particular flavor compounds, using agar plates and a minimal medium (YNB) supplemented with glucose (2%) and TFL (0.6 mM) or cerulenin (6 µM).

Enzymatic activities were evaluated using qualitative assays. The activity of β-lyase was screened using a medium containing 0.1% S-methyl-l-cysteine, 0.01% pyridoxal-50-phosphate, 1.2% Yeast Carbon Base, and 2% agar, with pH adjusted to 3.5. β-glycosidase activity was tested using a medium containing 0.5% cellobiose (4-O-β-d-glucopyranosyl-d-glucose), 0.67% yeast nitrogen base, and 2% agar. Proteolytic activity was evaluated by spotting yeast strain on skim milk agar medium. Qualitative detection of biogenic amines (histamine, tyramine and putrescine) was performed using differential culture media containing yeast extract (3%), glucose (1%), the amino acid precursor (2%), histidine, tyrosine or ornithine, and bromocresol purple (0.015 g.L−1), at a final pH adjusted to 5.2. The potential ability to produce hydrogen sulfide (H2S) associated to sulfite reductase activity was evaluated by growing yeast cells on BiGGY agar.

2.3. Grape Juice and Inocula Preparation

Natural grape-juice was obtained by crushing grapes of the Vitis vinifera L. cv. Touriga Nacional; after homogenization, the juice was clarified by centrifugation at 12,734× g for 10 min (Sorvall centrifuge GSA 6-Place Rotor, Marshall Scientific, Hampton, NH 03842, USA) and was carefully separated from the solid fraction. A sample of the grape-juice was collected at this point for routine analysis (Table1). After pasteurization at 70◦

C for 10 min, the grape-juice was immediately cooled on ice. For each strain, the inoculum was prepared by separately pre-growing the yeast cells in 50 mL-flasks, containing 25 mL of synthetic grape-juice medium (GJM), original recipe of [69] with minor modifications in the nitrogen composition. Nitrogen was added up to 267 mg YAN/L, supplied as di-ammonium phosphate (DAP). The flasks were incubated overnight at 25◦

C in an orbital shaker (IKA KS 4000 ic Control, VWR International, Radnor, PA 19087-8660, USA) set at 150 rpm.min−1. Both strains were inoculated in grape-juice with an initial cellular concentration of 106cfu·mL−1.

2.4. Fermentation Trials

Fermentations trials were conducted by inoculating (1) a single culture of S. ludwigii UTAD17 (Sl), (2) a single culture of S. cerevisiae (Sc), (3) a mixed culture of S. ludwigii UTAD17 and S. cerevisiae (Sl+Sc) inoculated simultaneously, prior to fermentation, or (4) a mixed culture in which S. cerevisiae was inoculated sequentially, 72 h after S. ludwigii UTAD17 (Sl_Sc). Single and mixed culture fermentations were carried out in duplicates and triplicates, respectively, using a previously described system [70] consisting of 100 mL flasks filled to 2/3 of their volume (80 mL) and fitted with a side-arm port sealed with a rubber septum for anaerobic sampling. Two flasks containing uninoculated grape-must were used as control. The flasks were maintained at 25◦C under static conditions. Fermentations were monitored daily by weight loss as an estimation of CO2production and were allowed to proceed until no further weight loss was observed. For the assessment of growth parameters and analytical determinations, aseptic sampling was performed using a syringe-type system. After fermentation, the wines were centrifuged (10 min at 5500 rpm, Sigma 3-18K refrigerated Centrifuge, 37520 Osterode

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am Harz, DE) to remove yeast cells and were kept at −20◦C until the analytical determinations were performed.

2.5. Determination of Growth and Fermentation Parameters

Growth kinetics were monitored by viable cell plate counting (cfu·mL−1) on YPD agar or Lysine agar medium plates incubated at 28◦C for 48–72 h. The lysine agar medium was used to directly assess S. ludwigii UTAD17 viability in mixed-culture fermentations, since S. cerevisiae is unable to grow in a culture medium in which lysine is the sole nitrogen source [71]. The maximum fermentation rate (MaxFR) was determined from the slope of the linear dependence of the steepest incline in weight (g) at the corresponding time points (h), and fermentation purity (FP) was determined as acetic acid (g L−1)/ethanol (%v:v−1).

2.6. Analytical Determinations

The amount of glucose and fructose, acetic acid, as well as Yeast Assimilable Nitrogen (YAN), comprising primary amino nitrogen (PAN) and ammonium, were enzymatically determined using a Y15 autoanalyzer (Biosystems S.A, Barcelona, Spain). Total SO2, pH, and titratable acidity were determined according to the standard methods compiled in the Compendium of International Methods of Analysis of Musts and Wines [72].

Ethanol and glycerol concentrations were determined in a high-performance liquid chromatography system (HPLC Flexar, PerkinElmer, Shelton, Connecticut, EE. UU) equipped with the ion exclusion cation exchange column Aminex HPX-87H (Bio-Rad Laboratories, Hercules, CA, USA) and refractive index detector. The column was eluted using sulfuric acid (0.005 N) at 60◦C and a 0.6 mL/min flow rate. Samples were previously filtered through a membrane (Millipore, 0.22 µm pore size) before an injection of 6 µL. The components were identified through their relative retention times, compared to the respective standards, using the Perkin Elmer Chromera Software.

Aliphatic higher alcohols (1-propanol, 1-butanol, 2-methyl-1-butanol and 3-methyl-1-butanol), acetaldehyde, and ethyl acetate were analyzed as described Moreira et al. [73] by using a Hewlett-Packard 5890 (Hewlett-Packard, Palo Alto, CA 94304, USA) gas chromatograph equipped with a flame ionization detector (GC-FID) and connected to a H.P. 3396 Integrator. Fifty microliters of 4-methyl-2- pentanol at 10 g L−1was added to 5 mL of wine as the internal standard. The sample (1 µL) was injected (split, 1: 30) into a CP-WAX 57 CB column (Chrompack) of 50 m × 0.25 mm and 0.2 µm phase thickness. The program temperature varied from 40◦C (10 min) to 80◦C (10 min) at 3◦C min−1and from 80◦C to 200◦C (4 min) at 15◦C min−1. Injector and detector temperatures were set at 220◦C. Carrier gas was H2at 1–2 mL min−1.

The determination of 2-phenylethanol, acetates of higher alcohols (isoamyl acetate, 2-phenylethyl acetate) and ethyl esters of fatty acids (ethyl butanoate, ethyl hexanoate and ethyl octanoate), volatile fatty acids (butyric, isobutyric, isovaleric acids) and free fatty acids (hexanoic, octanoic and decanoic acids) was performed in a Hewlett Packard 5890 gas chromatograph, equipped with a flame ionization detector. For this purpose, 50 mL of wine, with 4-decanol at 1.5 mg/L as the internal standard, was extracted successively with 4, 2, and 2 mL of ether–hexane (1:1 v:v−1) for 5 min. The organic phase (1 µL) was injected (splitless) into a BP21 (SGE) column of 50 m × 0.22 mm and 0.25 µm phase thickness. The temperature program was 40◦

C (1 min) to 220◦

C (15 min), at 2◦

C·min−1_{. Injector and detector} temperatures were set at 220◦C. The carrier gas used was H2at 1–2 mL min−1.

2.7. Statistical Analysis

The data are presented as mean values with their standard deviation. One-way analysis of variance (ANOVA) of the inoculation strategy on yeast growth, fermentation activity, and volatile and non-volatile compounds was performed using the JMP 7.0 software (SAS Inc., 2007). If significant differences were found with ANOVA (p < 0.05), then Student’s t-test was used for the paired comparisons. Partial least squares linear discriminant analysis (PLS–DA) was performed to

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discriminate the wines, based on volatile and non-volatile compounds, using the MATLAB R2018b environment (The MathWorks Inc.; version 9.5.; Natick, MA, USA) state abbreviation). All data were previously standardized.

2.8. Comparison of S. ludwigii UTAD17 ‘ORFeome’ with S. cerevisiae s288c

Recently S. ludwigii UTAD17 has been sequenced and annotated with the predicted set of open reading frames (ORFeome) estimated to be about 4015 protein-coding genes [67]. This whole-genome shotgun is available in the European Nucleotide Archive (ENA) under the accession number UFAJ01000000 (contigs UFAJ01000001 through UFAJ01001360; study accession number PRJEB27462; read accession number SAMEA4945973). Herein, a supervised analysis was performed by BLASTp using the proteomes of S. ludwigii UTAD17 and S. cerevisiae S288c, looking for the presence or absence of protein-coding genes that could underlie the observed physiological traits. S. ludwigii UTAD17 proteins were considered similar to those present in S. cerevisiae S288c when the resulting alignment had an associated e-value below e−20and a minimum identity of 30% (Table S1).

3. Results and Discussion

3.1. Phenotypic Characterization of S. ludwigii UTAD17

In order to assess the potential of S. ludwigii UTAD17 to be used in winemaking, a phenotypic profiling was performed for a number of oenological traits, as determined by the International Organisation of Vine and Wine (OIV) [68] (Figure S1). Ethanol is the main metabolite produced during wine fermentation while SO2and copper are applied by winemakers as antimicrobial agents to control spoilage in wineries and vineyards, respectively. These compounds have recognized negative effects on yeast growth and fermentative activity which could lead to stuck and sluggish fermentations [74]. The results obtained showed that S. ludwigii UTAD17 displayed high resistance to SO2(4 mM), ethanol (12% v:v−1_{), and copper (2 mM) (Figure S1). The ability to produce biogenic animes which are toxic} to humans, [21] was also evaluated. Interestingly, S. ludwigii UTAD17 did not present decarboxylase activities responsible for the production of histamine, tyramine and putrescine. On the other hand, S. ludwigii UTAD17 exhibited β-glucosidase and β-lyase activities involved in the liberation of terpenes from glycosylated precursors [12] and volatile thiols from cysteinylated precursors [19]. In line with the results obtained, S. ludwigii UTAD17 presents important features for a wine yeast starter, since it is able to adjust to winemaking stress, can contribute to the improvement of wine aromatic profile and does not compromise consumers’ health.

3.2. Yeast Growth Kinetics and Fermentation Profiles

To evaluate the performance of S. ludwigii UTAD17 in winemaking conditions, fermentations were conducted either in single culture or in consortium with the commercial wine strain Saccharomyces cerevisiae QA23, inoculated simultaneously or sequentially, at 72 h. In parallel, a control fermentation was carried out using Saccharomyces cerevisiae QA23 in single culture, for comparison.

The growth dynamics and fermentation profiles for each single and mixed culture trials are presented in Figure1. All fermentations were completed, although differences in the total time of fermentation were observed. The S. ludwigii UTAD17 showed a sugar uptake preference similar to S. cerevisiae strains, consuming glucose more rapidly than fructose. While in pure culture, S. ludwigii UTAD17 displayed a significantly lower fermentation rate than S. cerevisiae, although it was able to ferment the grape must sugar to dryness (below 4 g L−1) within 11 days, six more days than the time required for the high fermenter strain S. cerevisiae Lalvin QA23 (Table1and Figure1B). This lower fermentative activity of S. ludwigii UTAD17 was not attributable to the differences in the biomass, which is known to have a great influence in determining the fermentation activity [69,75]. Indeed, both species, inoculated at the same amount (1 × 106cfu·mL−1), resumed growth almost immediately after inoculation and, albeit with differences in the growth rate (Figure1A), achieved similar

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maximum cell populations—1.2 × 108cfu·mL−1for S. cerevisiae (after 48 h) and 1.1 × 108cfu·mL−1 for S. ludwigii UTAD17 (after 72 h). A low fermentative capacity together with a high susceptibility to ethanol is believed to underlie the reduced competitiveness of non-Saccharomyces species along wine fermentation [76]. The phenotypic screening performed showed that S. ludwigii UTAD17 is able to tolerate up to 12% (v:v−1) of ethanol (Figure S1), which is above the 10.2–10.4% (v:v−1) achieved in the final wines, indicating that the decline in cell viability registered in the later stage of the fermentation should result from other factors. The predicted ORFeome of S. ludwigii UTAD17 showed that this species is equipped with enzymes required for ethanol production from glucose, including hexoses transporters, glycolytic enzymes, and alcohol dehydrogenases (Table S1). Recently, genomic sequencing of an H. guilliermondii wine strain revealed that one of the key factors contributing to the reduced fermentation ability of this species is the lack of genes for the biosynthesis of thiamine [77], a cofactor of the pyruvate decarboxylase enzyme that is known to play an essential role in determining the regulation of the glycolytic flux [78,79]. This is not apparently the case in S. ludwigii UTAD17, since the thiamine-biosynthesis genes could be predicted from the genomic sequence of the UTAD17 strain (Table S1) [67]. Further studies are required to understand the lower fermentation rate exhibited by the S. ludwigii UTAD17, in comparison with S. cerevisae, one of the possibilities being a low activity of critical glycolytic enzymes, as recently shown to be the case in H. uvarum pyruvate kinase [80]. Contrary to that observed for S. cerevisiae which almost entirely consumed the nitrogen available in the medium, S. ludwigii UTAD17 displayed a preferential consumption of amino acids (PAN) over ammonium and left about 60 mg/L of YAN in the final wine (Table1). It has been proposed that differences in the efficiency of nitrogen consumption, in general, and in the ability to uptake specific nitrogen sources from the grape-must account for variations in the fermentative activity of S. cerevisiae strains [81,82]. In this context, it would be interesting to determine whether the differences observed in the fermentation performances of S. cerevisiae and S. ludwigii UTAD17 (Figure1) are the result of differences in their nitrogen uptake capability (Table1).

Table 1.Physicochemical composition of initial grape-must and wines obtained by single-cultures of S. ludwigii UTAD17 (Sl) and S. cerevisiae QA23 (Sc) or in consortium—mixed simultaneously (Sl+Sc) and sequentially (Sl_Sc) in natural grape-juice of Vitis vinifera L. cv. Touriga Nacional at 25◦C under static conditions. Compound Grape-Must Sl Sc Sl+Sc Sl_Sc Sugars (g L−1) 182.140 ± 3.62 2.273 ± 0.733a 0.328 ± 0.284b 0.190 ± 0.242b 0.018 ± 0.009b Ethanol (% v:v−1₎ _- _{10.195 ± 0.194}a _{10.391 ± 0.025}a _{10.201 ± 0.077}a _{10.355 ± 0.271}a Glycerol (g L−1₎ _- _{6.279 ± 0.024}c _{7.671 ± 0.060}ab _{7.659 ± 0.302}a _{6.800 ± 0.658}bc Acetic Acid (g L−1₎ _- _{0.138 ± 0.004}bc _{0.170 ± 0.018}a _{0.149 ± 0.004}ab _{0.120 ± 0.011}c Titratable Acidity (g L−1) 8.010 ± 0.350 8.100 ± 0.120ab 7.980 ± 0.000a _{8.390 ± 0.010}a _{7.690 ± 0.130}b Total SO2(mg L−1) - 14.830 ± 0.430a 15.300 ± 0.820a 15.360 ± 0.000a 16.380 ± 3.020a pH 2.990 ± 0.011 2.957 ± 0.003a 2.926 ± 0.031b _{2.961 ± 0.000}a _{2.956 ± 0.004}a YAN (mg L−1₎ _{196.869 ± 2.339} _{61.214 ± 5.029}a _{5.250 ± 0.354}c _{4.000 ± 0.000}c _{22.594 ± 6.731}b YAN(72 h)(mg L−1) - 79.584 ± 1.996a 12.250 ± 3.889b 13.168 ± 0.289b 68.418 ± 19.340a PAN (mg L−1₎ _{92.000 ± 9.899} _{20.500 ± 2.121}a _{5.250 ± 0.354}b _{4.000 ± 0.000}b _{19.167 ± 4.537}a PAN(72 h)(mg L−1) - 29.000 ± 1.414a 12.250 ± 3.889b 13.168 ± 0.289b 26.333 ± 4.646a NH4(mg L−1) 127.500 ± 9.192 49.500 ± 3.536a ndc ndc 4.170 ± 2.843b NH4(72 h)(mg L−1) - 61.500 ± 0.707a ndb ndb 51.167 ± 17.905a Sugars 72 h (g L−1₎ _- _{109.252 ± 1.555}a _{41.174 ± 0.246}b _{42.883 ± 2.115}b _{101.200 ± 11.232}a MaxFR (g of CO2 Vh−1). - 0.051 ± 0.004b 0.100 ± 0.005a 0.098 ± 0.002a 0.058 ± 0.006b FP - 0.014 ± 0.001a,b 0.017 ± 0.002a _{0.015 ± 0.001}a _{0.011 ± 0.002}b Data are expressed as triplicate means for mixed trials and duplicate means for single culture trials ± standard deviations. Values in the same row with different superscript letters are significantly different (p < 0.05). YAN—yeast assimilable nitrogen. PAN—primary amino nitrogen. MaxFR—maximum fermentation rate. FP—fermentation purity (acetic acid (g L−1)/ ethanol (% v:v−1)). nd—not detected. -: not measured.

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Microorganisms 2019, 7, 478 7 of 16 Microorganisms 2019, 7, x FOR PEER REVIEW

Figure 1. Means ± standard deviations of (A) yeast cell counts of S. ludwigii UTAD17 (green squares) and S. cerevisiae QA23 (red triangles) in single and mixed cultures; and

(B) Fermentation profiles (diamonds), glucose (filled circles), and fructose (clear circles) concentrations during single and mixed culture trials.

Figure 1.Means ± standard deviations of (A) yeast cell counts of S. ludwigii UTAD17 (green squares) and S. cerevisiae QA23 (red triangles) in single and mixed cultures; and (B) Fermentation profiles (diamonds), glucose (filled circles), and fructose (clear circles) concentrations during single and mixed culture trials.

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When the two strains were co-inoculated simultaneously (Sc+Sl), a decrease in both strain populations was noted to likely reflect the more competitive environment in terms of space [83] and nutrients, two factors that were previously found to determine yeast–yeast interactions [32,74]. Yet, S. cerevisiae dominated over S. ludwigii UTAD17, in line with the notion that S. cerevisiae is better adapted to grape-juice per se than non-Saccharomyces yeasts [84]. Notably, although S. ludwigii UTAD17 was able to maintain a substantial viable population throughout these Sc+Sl fermentations, the rate and total time of fermentation was mostly similar, compared to S. cerevisiae in a single culture. In the sequential mixed-culture trials (Sl_Sc), S. ludwigii UTAD17 initiated fermentation, and S. cerevisiae QA23 was later inoculated at 72 h. At this stage, about 40% of the initial sugars were fermented by S. ludwigii UTAD17 and the assimilable nitrogen concentration was significantly lowered (Table1). Contrary to that observed in simultaneous fermentations, in this case, S. ludwigii UTAD17 dominated over S. cerevisiae. Indeed, growth of the non-Saccharomyces strain proceeded as in a single culture, exhibiting a similar growth rate and loss of viability. On the other hand, in these experiments S. cerevisiae QA23 growth was limited to a maximum population of about 1.1 × 107cfu mL−1, attained 24 h after its inoculation, most probably due to the low assimilable nitrogen available in the medium. Nevertheless, there was an increase in the fermentation rate and sequential fermentations were successfully completed in eight days, taking 72 h more than S. cerevisiae in single culture. Adjusting the YAN levels of the fermenting must at this stage, could be an option to improve the growth of S. cerevisiae and the fermentation performance.

3.3. Effect of S. ludwigii UTAD17 on Wine Composition and Aroma Profile

The primary physiochemical parameters of the wines obtained are presented in Table 1. The production of ethanol is an essential attribute to define the use of yeasts in the production of fermented beverages. S. ludwigii UTAD17 showed a similar efficiency of sugar-to-ethanol conversion to that of S. cerevisiae QA23, as the ethanol levels of the final wines, which ranged from 10.2 to 10.4% (v:v−1), were not significantly different. Likewise, no significant differences were found on the amount of SO2formed in each fermentation. Both strains produced up to 20 mg mg L−1and thus they were considered low-sulfite-forming yeasts [85]. Overall, all fermentations resulted in lower levels of acetic acid. Slightly lower levels of acetic acid were found in wines where S. ludwigii UTAD17 was involved, as compared to the wines only fermented by S. cerevisiae and significantly lower levels of this compound were detected on the sequentially inoculated wines (Table1). These are promising features since the amount of both metabolites is tightly limited by regulations, might depreciate wine aroma (especially acetic acid) or raise concerns about consumers’ safety (SO2) and, thus, they should be kept at the lowest possible levels. The lower acetic acid produced in the fermentations dominated by the non-Saccharomyces yeast was accompanied by significantly lower levels of glycerol in all fermentations, suggesting that S. ludwigii UTAD17 management strategy for NADH/NAD+_{recycling and maintenance} of redox balance is similar to that of S. cerevisiae [86]. This connection between acetic acid and glycerol production is not as clear in other non-Saccharomyces. For instance, in single culture fermentations, Starmerella bacillaris appears to be a high glycerol and low acetic acid producer [53], while H. uvarum seems to be a low glycerol and high acetic acid producer of yeast [87].

In order to evaluate how S. ludwigii UTAD17 affected the final aroma composition, the different wines were analyzed by gas chromatography. Eighteen yeast-derived aroma compounds were quantified, five alcohols, six acids, four ethyl-esters, two acetates, and one aldehyde (Table2). Those compounds that were found to be significantly different (p < 0.05), along with glycerol and acetic acid (Table1), were used for Partial Least Squares–Discriminant Analysis (PLS–DA), in order to distinguish the wines obtained with the different inoculation strategies (Figure2). The first component accounted for 69.99%, while component 2 explained 18.83% of the total variation. Replicate experiments were well grouped on the PLS. A clear separation was observed between the wines whose fermentation were dominated by S. cerevisiae, and those governed by S. ludwigii UTAD17. Although S. ludwigii UTAD17 produced overall significantly lower levels of volatile compounds (Table2), it should be highlighted

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that the wines obtained by simultaneous co-inoculation of both strains (Sl+Sc), located in the upper-left quadrant (Figure2), were characterized by a greater diversity of flavors and complexity, as compared with those fermented by S. cerevisiae alone. Overall, fermentations conducted by S. ludwigii UTAD17 resulted in wines characterized by higher levels of 1-butanol and butyric and isobutyric acids, which were found to be 2 to 3 times higher than that in Sc and Sc+Sl wines (Table2). 1-butanol and the short-chain fatty acid, butyric acid, are synthetized from 2-ketovalerate following decarboxylation to the aldehyde precursor, butyraldehyde, which is either reduced or oxidized, respectively [88]. In this study we could only speculate that the high levels of both compounds produced by S. ludwigii UTAD17 could result from either an excess of the intermediate α-keto acid or the use of this redox duality of the last steps of the Ehrlich pathway to help maintain the redox balance of the cell [89].Microorganisms 2019, 7, x FOR PEER REVIEW

Figure 2. Partial least squares-discriminant analysis (PLS–DA) plot of wines obtained with the different inoculation strategies using volatile and non-volatile compounds that were significantly different among treatments—single-culture of S. ludwigii UTAD17(Sl) and S. cerevisiae (Sc) or in consortium—mixed simultaneously (Sl+Sc) and sequentially (Sl_Sc).

Overall, the above-described results gave good insights on the potential use of S. ludwigii UTAD17 in winemaking. Given the low fruity/estery nature of this strain, it could be a good option to obtain wines with a greater expression of grape varietal characteristics. Nowadays, winemakers make use of a blend of wines fermented with different yeast strains/species or different grape varieties to create wines tailored to meet consumer expectations. Following this line of winemaking, and considering the trend of the use of non-Saccharomyces yeasts to obtain wines with distinct aroma profiles, it should be underlined that the aromatic characteristics of S. ludwigii, UTAD17, which greatly differs from the traditional S. cerevisiae, might confer a peculiar imprint onto the final product. Nevertheless, our results warrant further studies to evaluate whether the observed differences in chemical composition can be perceived during sensory evaluation.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Phenotypic profile of the S. ludwigii UTAD17; Table S1: Comparative analysis of the predicted S. ludwigii UTAD17 ORFeome with sets of proteins of S. cerevisiae S288c

Author Contributions: A.M.-F., N.P.M., and C.B. conceived and designed the experiments; M.E. and C.B. performed the experiments; I.V. performed the GC analyses; M.J.T. and M.E. performed the bioinformatics analysis. All authors analyzed the data; M.E. drafted the manuscript; All authors revised and approved the submitted version.

Funding: This work was funded by European Regional Development Fund (ERFD) through POCI-COMPETE 2020 and by FCT through the project SMARTWINE—Smarter wine fermentations: integrating OMICS tools (PTDC/AGR-TEC/3315/2014, POCI-01-0145-FEDER-016834). Support received from INTERACT project no. NORTE-01-0145-FEDER-000017 through its line of research entitled VitalityWINE, and from INNOVINE&WINE. NORTE-01-0145-FEDER-000038, co-financed by ERDF through NORTE 2020 is also

Figure 2.Partial least squares-discriminant analysis (PLS–DA) plot of wines obtained with the different inoculation strategies using volatile and non-volatile compounds that were significantly different among treatments—single-culture of S. ludwigii UTAD17(Sl) and S. cerevisiae (Sc) or in consortium—mixed simultaneously (Sl+Sc) and sequentially (Sl_Sc).

Previous studies have put aside the use of S. ludwigii in the winemaking industry, given the large amounts of ethyl acetate and acetaldehyde produced by these strains [7,8,61]. Notably, the S. ludwigii UTAD17 strain is, apparently, a low producer of both compounds as the levels detected were significantly inferior to those obtained in both Sc and Sl+Sc fermentations. Additionally, none-to-low levels of acetate and ethyl esters, which are responsible for the pleasant fruity and floral bouquet of wines, were found to be produced by S. ludwigii UTAD17. The predicted UTAD17 ORFeome did not include proteins similar to the S. cerevisiae acetyl transferases ScATF1, ScATF2, and ScAYT1, required for synthesis of acetate esters (Table S1). As such, the minor amounts of these compounds found on Sl_Sc fermentations was likely attributable to S. cerevisiae QA23 activity. Contrarily, we found that S. ludwigii UTAD17 harbors proteins similar to S. cerevisiae ethanol acyl-coA transferases ScEHT1 and ScEEB1, responsible for ethyl ester synthesis through the condensation between ethanol and fatty acyl-CoA [90], and ScIAH1 involved in esters hydrolysis [91]. In this case, the limited production of ethyl esters by S. ludwigii UTAD17 might result from differences in the activity levels of these enzymes. A more in-depth analysis of this genomic information is being undertaken that will shed light on the molecular foundations underlying some of the intriguing physiological traits of this yeast such as high SO2resistance, lower fermentative power, and wine aroma. From a practical point of view, this information will be very useful for a rational application of this strain, depending on the winemaking conditions

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Table 2.Concentration of volatile compounds detected and quantified by Gas Chromatography equipped with Flame ionization Detector (GC–FID) in wine obtained by a single-culture of S. ludwigii UTAD17(Sl) and S. cerevisiae (Sc) or in consortium—mixed simultaneous (Sl+Sc) and sequential (Sl_Sc), in natural grape-juice of Vitis vinifera L. cv. Touriga Nacional at 25◦C, under static conditions.

Compound (mg L−1₎ _Sl _Sc _Sl+Sc _{Sl_Sc} _{OT (mg/L)} _OD

Alcohols

1-propanol 14.315 ± 0.402c _{44.383 ± 1.298}a _{43.985 ± 0.516}a _{17.934 ± 1.557}b _306.000 _{Alcohol, ripe fruit}

1-butanol 35.428 ± 1.045a 15.040 ± 0.220c 15.335 ± 1.428c 30.792 ± 0.316b 150.000 Medicinal 2-Methyl-1-butanol 17.882 ± 1.033ab _{19.040 ± 0.721}a _{18.966 ± 1.087}a _{15.123 ± 0.436}b _30.000 _{Alcohol, nail polish}

3-Methyl-1-butanol 74.963 ± 6.858b 102.984 ± 3.465a 101.176 ± 1.794a 69.184 ± 1.217b 30.000 Whiskey, nail polish 2-Phenylethanol 18.945 ± 0.728b 27.040 ± 1.881a _{26.997 ± 1.398}a _{16.869 ± 1.015}b _14.000 _{Rose, honey}

Table 2. Concentration of volatile compounds detected and quantified by Gas Chromatography equipped with Flame ionization Detector (GC–FID) in wine obtained by a single-culture of S. ludwigii UTAD17(Sl) and S. cerevisiae (Sc) or in consortium—mixed simultaneous (Sl+Sc) and sequential (Sl_Sc), in natural grape-juice of Vitis vinifera L. cv. Touriga Nacional at 25 °C, under static conditions.

Compound (mg L-1) Sl Sc Sl+Sc Sl_Sc OT (mg/L) OD

Alcohols

1-propanol 14.315 ± 0.402 c _{44.383 ± 1.298}a _{43.985 ± 0.516}a _{17.934 ± 1.557}b _306.000 _{Alcohol, ripe fruit}

1-butanol 35.428 ± 1.045 a _{15.040 ± 0.220}c _{15.335 ± 1.428}c _{30.792 ± 0.316}b _150.000 _Medicinal

2-Methyl-1-butanol 17.882 ± 1.033 ab _{19.040 ± 0.721}a _{18.966 ± 1.087}a _{15.123 ± 0.436}b _30.000 _{Alcohol, nail polish}

3-Methyl-1-butanol 74.963 ± 6.858 b _{102.984 ± 3.465}a _{101.176 ± 1.794}a _{69.184 ± 1.217}b _30.000 _{Whiskey, nail polish}

2-Phenylethanol 18.945 ± 0.728 b _{27.040 ± 1.881}a _{26.997 ± 1.398}a _{16.869 ± 1.015}b _14.000 _{Rose, honey}

⅀ 161.534 ± 8.609 b _{208.486 ± 3.824}a _{206.459 ± 6.122}a _{149.962 ± 4.092}b

Acetate Esters

Phenylethyl Acetate ndb _{0.181 ± 0.011}a _{0.177 ± 0.024}a _ndb _0.250 _Flowery

Isoamyl Acetate ndc _{0.807 ± 0.106}a _{0.725 ± 0.155}a _{0.171 ± 0.120}b _0.030 _Banana

⅀ ndc _{0.988 ± 0.117}a _{0.902 ± 0.163}a _{0.171 ± 0.120}b

Ethyl Esters

Ethyl Acetate 32.679 ± 6.895 b _{40.995 ± 0.393}ab _{47.392 ± 7.071}a _{28.387 ± 5.742}b _7.500 _{Fruity, vinegar, nail polish, acetic}

Ethyl Butanoate ndb _{0.604 ± 0.182}a _{0.630 ± 0.066}a _{0.669 ± 0.118}a _0.020 _{Apple, strawberry, fruity}

Ethyl Hexanoate ndd _{0.301 ± 0.012}a _{0.231 ± 0.024}b _{0.082 ± 0.020}c _0.005 _{Green apple, fruity}

Ethyl Octanoate ndb _{0.312 ± 0.046}a _{0.410 ± 0.080}a _ndb _0.002 _{Pear, fruity}

⅀ (except ethyl acetate)

ndc _{1.216 ± 0.149}a _{1.271 ± 0.160}a _{0.752 ± 0.132}b

Fatty Acids

Isobutyric Acid 2.430 ± 0.072 a _{1.077 ± 0.194}b _{1.242 ± 0.205}b _{2.494 ± 0.172}a _2.300 _Fatty

Butyric Acid 1.892 ± 0.074 a _{0.631 ± 0.089}b _{0.726 ± 0.056}b _{1.880 ± 0.074}a _10.000 _{Fatty, rancid}

Isovaleric Acid ndb _{0.231 ± 0.040}a _{0.251 ± 0.045}a _ndb _0.033 _{Fatty, rancid}

Hexanoic Acid 0.591 ± 0.023 c _{1.435 ± 0.064}a _{1.350 ± 0.027}b _{0.558 ± 0.034}c _0.420 _{Cheese, fatty}

Octanoic Acid 0.727 ± 0.175 c _{2.383 ± 0.060}a _{1.559 ± 0.127}b _{0.710 ± 0.145}c _0.500 _{Fatty, unpleasant}

Decanoic Acid 0.222 ± 0.005 a _{0.224 ± 0.031}a _ndb _ndb _1.000 _{Fat, rancid}

⅀ 5.861 ± 0.293 a _{5.980 ±0.475}a _{5.127 ± 0.145}b _{5.642 ± 0.329}ab

Acetaldehyde 20.279 ± 0.472 b _{25.328 ± 1.400}a _{25.661 ± 1.500}a _{25.944 ± 2.672}a _10.000 _{Sherry, nutty, bruised apple} Data are expressed as means ± standard deviations resulting from triplicate experiments for mixed trials and duplicates for single culture trials. OT—odor threshold; OD—odor descriptions. Odor thresholds and odor descriptions can be found in the literature [92–98]. Values in the same row with different superscript letter are significantly different (p < 0.05). nd—not detected.

161.534 ± 8.609b _{208.486 ± 3.824}a _{206.459 ± 6.122}a _{149.962 ± 4.092}b

Acetate Esters

Phenylethyl Acetate ndb 0.181 ± 0.011a _{0.177 ± 0.024}a _ndb _0.250 _Flowery

Isoamyl Acetate ndc 0.807 ± 0.106a 0.725 ± 0.155a 0.171 ± 0.120b 0.030 Banana Microorganisms 2019, 7, x FOR PEER REVIEW

Compound (mg L-1₎ _Sl _Sc _Sl+Sc _{Sl_Sc} _{OT (mg/L)} _OD

Alcohols

⅀ 161.534 ± 8.609 b _{208.486 ± 3.824}a _{206.459 ± 6.122}a _{149.962 ± 4.092}b

Acetate Esters

⅀ ndc _{0.988 ± 0.117}a _{0.902 ± 0.163}a _{0.171 ± 0.120}b

Ethyl Esters

ndc _{1.216 ± 0.149}a _{1.271 ± 0.160}a _{0.752 ± 0.132}b

Fatty Acids

⅀ 5.861 ± 0.293 a _{5.980 ±0.475}a _{5.127 ± 0.145}b _{5.642 ± 0.329}ab

ndc 0.988 ± 0.117a 0.902 ± 0.163a 0.171 ± 0.120b Ethyl Esters

Ethyl Acetate 32.679 ± 6.895b 40.995 ± 0.393ab 47.392 ± 7.071a 28.387 ± 5.742b 7.500 Fruity, vinegar, nail polish, acetic Ethyl Butanoate ndb 0.604 ± 0.182a 0.630 ± 0.066a 0.669 ± 0.118a 0.020 Apple, strawberry, fruity Ethyl Hexanoate ndd _{0.301 ± 0.012}a _{0.231 ± 0.024}b _{0.082 ± 0.020}c _0.005 _{Green apple, fruity}

Ethyl Octanoate ndb 0.312 ± 0.046a _{0.410 ± 0.080}a _ndb _0.002 _{Pear, fruity}

Microorganisms 2019, 7, x FOR PEER REVIEW

Alcohols

⅀ 161.534 ± 8.609 b _{208.486 ± 3.824}a _{206.459 ± 6.122}a _{149.962 ± 4.092}b

Acetate Esters

⅀ ndc _{0.988 ± 0.117}a _{0.902 ± 0.163}a _{0.171 ± 0.120}b

Ethyl Esters

ndc _{1.216 ± 0.149}a _{1.271 ± 0.160}a _{0.752 ± 0.132}b

Fatty Acids

⅀ 5.861 ± 0.293 a _{5.980 ±0.475}a _{5.127 ± 0.145}b _{5.642 ± 0.329}ab

(except ethyl

acetate) ndc 1.216 ± 0.149a _{1.271 ± 0.160}a _{0.752 ± 0.132}b

Fatty Acids

Isobutyric Acid 2.430 ± 0.072a 1.077 ± 0.194b 1.242 ± 0.205b 2.494 ± 0.172a 2.300 Fatty Butyric Acid 1.892 ± 0.074a 0.631 ± 0.089b _{0.726 ± 0.056}b _{1.880 ± 0.074}a _10.000 _{Fatty, rancid}

Isovaleric Acid ndb 0.231 ± 0.040a 0.251 ± 0.045a ndb 0.033 Fatty, rancid

Hexanoic Acid 0.591 ± 0.023c _{1.435 ± 0.064}a _{1.350 ± 0.027}b _{0.558 ± 0.034}c _0.420 _{Cheese, fatty}

Octanoic Acid 0.727 ± 0.175c 2.383 ± 0.060a 1.559 ± 0.127b 0.710 ± 0.145c 0.500 Fatty, unpleasant

Decanoic Acid 0.222 ± 0.005a 0.224 ± 0.031a ndb _ndb _1.000 _{Fat, rancid}

Microorganisms 2019, 7, x FOR PEER REVIEW

Alcohols

⅀ 161.534 ± 8.609 b _{208.486 ± 3.824}a _{206.459 ± 6.122}a _{149.962 ± 4.092}b

Acetate Esters

⅀ ndc _{0.988 ± 0.117}a _{0.902 ± 0.163}a _{0.171 ± 0.120}b

Ethyl Esters

ndc _{1.216 ± 0.149}a _{1.271 ± 0.160}a _{0.752 ± 0.132}b

Fatty Acids

⅀ 5.861 ± 0.293 a _{5.980 ±0.475}a _{5.127 ± 0.145}b _{5.642 ± 0.329}ab

5.861 ± 0.293a _{5.980 ±0.475}a _{5.127 ± 0.145}b _{5.642 ± 0.329}ab

Acetaldehyde 20.279 ± 0.472b _{25.328 ± 1.400}a _{25.661 ± 1.500}a _{25.944 ± 2.672}a _10.000 _{Sherry, nutty, bruised apple}

Data are expressed as means ± standard deviations resulting from triplicate experiments for mixed trials and duplicates for single culture trials. OT—odor threshold; OD—odor descriptions. Odor thresholds and odor descriptions can be found in the literature [92–98]. Values in the same row with different superscript letter are significantly different (p < 0.05). nd—not detected.

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Overall, the above-described results gave good insights on the potential use of S. ludwigii UTAD17 in winemaking. Given the low fruity/estery nature of this strain, it could be a good option to obtain wines with a greater expression of grape varietal characteristics. Nowadays, winemakers make use of a blend of wines fermented with different yeast strains/species or different grape varieties to create wines tailored to meet consumer expectations. Following this line of winemaking, and considering the trend of the use of non-Saccharomyces yeasts to obtain wines with distinct aroma profiles, it should be underlined that the aromatic characteristics of S. ludwigii, UTAD17, which greatly differs from the traditional S. cerevisiae, might confer a peculiar imprint onto the final product. Nevertheless, our results warrant further studies to evaluate whether the observed differences in chemical composition can be perceived during sensory evaluation.

Supplementary Materials: The following are available online athttp://www.mdpi.com/2076-2607/7/11/478/s1, Figure S1: Phenotypic profile of the S. ludwigii UTAD17; Table S1: Comparative analysis of the predicted S. ludwigii UTAD17 ORFeome with sets of proteins of S. cerevisiae S288c

Author Contributions: A.M.-F., N.P.M., and C.B. conceived and designed the experiments; M.E. and C.B. performed the experiments; I.V. performed the GC analyses; M.J.T. and M.E. performed the bioinformatics analysis. All authors analyzed the data; M.E. drafted the manuscript; All authors revised and approved the submitted version.

Funding:This work was funded by European Regional Development Fund (ERFD) through POCI-COMPETE 2020 and by FCT through the project SMARTWINE—Smarter wine fermentations: integrating OMICS tools (PTDC/AGR-TEC/3315/2014, POCI-01-0145-FEDER-016834). Support received from INTERACT project no. NORTE-01-0145-FEDER-000017 through its line of research entitled VitalityWINE, and from INNOVINE&WINE. NORTE-01-0145-FEDER-000038, co-financed by ERDF through NORTE 2020 is also acknowledged. Support from FCT to BioISI (FCT/UID/Multi/04046/2019) and iBB (through contract UID/BIO/04565/ 2013) is also acknowledged. Acknowledgments: The authors thank Veronique Gomes for assistance in the statistical analysis and Rogério Tenreiro for the critical reading of the manuscript.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

1. Fleet, G.H.; Heard, G.M. Yeast growth during fermentation. In Wine Microbiology and Biotechnology (Fleet GH); Harwood Academic: Chur, Switzerland, 1993; pp. 27–54.

2. Egli, C.M.; Edinger, W.D.; Mitrakul, C.M.; Henick-Kling, T.; Henick-Kling, T. Dynamics of indigenous and inoculated yeast populations and their effect on the sensory character of Riesling and Chardonnay wines. J. Appl. Microbiol. 1998, 85, 779–789. [CrossRef] [PubMed]

3. Henick-Kling, T.; Edinger, W.; Daniel, P.; Monk, P. Selective effects of sulfur dioxide and yeast starter culture addition on indigenous yeast populations and sensory characteristics of wine. J. Appl. Microbiol. 1998, 84, 865–876. [CrossRef]

4. Ciani, M.; Mannazzu, I.; Marinangeli, P.; Clementi, F.; Martini, A. Contribution of winery-resident Saccharomyces cerevisiae strains to spontaneous grape must fermentation. Antonie Leeuwenhoek Int. J. Gen. Mol. Microbiol. 2004, 85, 159–164. [CrossRef] [PubMed]

5. Scholl, C.M.; Morgan, S.C.; Stone, M.L.; Tantikachornkiat, M.; Neuner, M.; Durall, D.M. Composition of Saccharomyces cerevisiae strains in spontaneous fermentations of Pinot Noir and Chardonnay. Aust. J. Grape Wine Res. 2016, 22, 384–390. [CrossRef]

6. Boulton, R.B.; Singleton, V.L.; Bisson, L.F.; Kunkee, R.E. Microbiological Spoilage of Wine and its Control. In Principles and Practices of Winemaking; Chapman & Hall: London, UK, 1996; pp. 352–381.

7. Ciani, M.; Maccarelli, F. Oenological properties of non-Saccharomyces yeasts associated with wine-making. World J. Microbiol. Biotechnol. 1998, 14, 199–203. [CrossRef]

8. Domizio, P.; Romani, C.; Comitini, F.; Gobbi, M.; Lencioni, L.; Mannazzu, I.; Ciani, M. Potential spoilage non-Saccharomyces yeasts in mixed cultures with Saccharomyces cerevisiae. Ann. Microbiol. 2011, 61, 137–144. [CrossRef]

9. Fernández, M.; Ubeda, J.; Briones, A. Typing of non-Saccharomyces yeasts with enzymatic activities of interest in wine-making. Int. J. Food Microbiol. 2000, 59, 29–36. [CrossRef]

(12)

10. Jolly, N.P.; Varela, C.; Pretorius, I.S. Not your ordinary yeast: Non-Saccharomyces yeasts in wine production uncovered. FEMS Yeast Res. 2014, 14, 215–237. [CrossRef]

11. Maturano, Y.P.; Assaf, L.A.R.; Toro, M.E.; Nally, M.C.; Vallejo, M.; De Figueroa, L.I.C.; Combina, M.; Vazquez, F. Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation. Int. J. Food Microbiol. 2012, 155, 43–50. [CrossRef]

12. Mendes-Ferreira, A.; Clímaco, M.C.; Mendes-Faia, A. The role of non-Saccharomyces species in releasing glycosidic bound fraction of grape aroma components Ð a preliminary study. J. Appl. Microbiol. 2001, 91, 67–71. [CrossRef]

13. Rosi, I.; Vinella, M.; Domizio, P. Characterization of Beta-Glucosidase Activity in Yeasts of Enological Origin. J. Appl. Bacteriol. 1994, 77, 519–527. [CrossRef] [PubMed]

14. Strauss, M.; Jolly, N.; Lambrechts, M.; Van Rensburg, P. Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. J. Appl. Microbiol. 2001, 91, 182–190. [CrossRef] [PubMed]

15. Viana, F.; Gil, J.; Genoves, S.; Valles, S.; Manzanares, P. Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits. Food Microbiol. 2008, 25, 778–785. [CrossRef] [PubMed]

16. Comitini, F.; Gobbi, M.; Domizio, P.; Romani, C.; Lencioni, L.; Mannazzu, I.; Ciani, M. Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol. 2011, 28, 873–882. [CrossRef]

17. De Benedictis, M.; Bleve, G.; Tristezza, M.; Tufariello, M.; Grieco, F. An optimized procedure for the enological selection of non-Saccharomyces starter cultures. Antonie Leeuwenhoek Int. J. Gen. Mol. Microbiol. 2011, 99, 189–200. [CrossRef]

18. Domizio, P.; Romani, C.; Lencioni, L.; Comitini, F.; Gobbi, M.; Mannazzu, I.; Ciani, M. Outlining a future for non-Saccharomyces yeasts: Selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation. Int. J. Food Microbiol. 2011, 147, 170–180. [CrossRef] 19. Belda, I.; Ruiz, J.; Alastruey-Izquierdo, A.; Navascués, E.; Marquina, D.; Santos, A. Unraveling the enzymatic

basis of wine “Flavorome”: A phylo-functional study of wine related yeast species. Front. Microbiol. 2016, 7, 1–13. [CrossRef]

20. Berbegal, C.; Spano, G.; Tristezza, M.; Grieco, F.; Capozzi, V. Microbial Resources and Innovation in the Wine Production Sector. S. Afr. J. Enol. Vitic. 2017, 38, 156–166. [CrossRef]

21. Barbosa, C.; Lage, P.; Esteves, M.; Chambel, L.; Mendes-Faia, A.; Mendes-Ferreira, A. Molecular and Phenotypic Characterization of Metschnikowia pulcherrima Strains from Douro Wine Region. Fermentation 2018, 4, 8. [CrossRef]

22. Nisiotou, A.; Sgouros, G.; Mallouchos, A.; Nisiotis, C.-S.; Michaelidis, C.; Tassou, C.; Banilas, G. The use of indigenous Saccharomyces cerevisiae and Starmerella bacillaris strains as a tool to create chemical complexity in local wines. Food Res. Int. 2018, 111, 498–508. [CrossRef]

23. Binati, R.L.; Innocente, G.; Gatto, V.; Celebrin, A.; Polo, M.; Felis, G.E.; Torriani, S. Exploring the diversity of a collection of native non-Saccharomyces yeasts to develop co-starter cultures for winemaking. Food Res. Int. 2019, 122, 432–442. [CrossRef] [PubMed]

24. Zironi, R.; Romano, P.; Suzzi, G.; Battistutta, F.; Comi, G. Volatile metabolites produced in wine by mixed and sequential cultures of Hanseniaspora guilliermondii or Kloeckera apiculata and Saccharomyces cerevisiae. Biotechnol. Lett. 1993, 15, 235–238. [CrossRef]

25. Ciani, M.; Fatichenti, F. Selective sugar consumption by apiculate yeasts. Lett. Appl. Microbiol. 1999, 28, 203–206. [CrossRef] [PubMed]

26. Moreira, N.; Mendes, F.; De Pinho, P.G.; Hogg, T.; Vasconcelos, I. Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must. Int. J. Food Microbiol. 2008, 124, 231–238. [CrossRef] [PubMed]

27. Andorrà, I.; Berradre, M.; Mas, A.; Esteve-Zarzoso, B.; Guillamón, J.M. Effect of mixed culture fermentations on yeast populations and aroma profile. LWT Food Sci. Technol. 2012, 49, 8–13. [CrossRef]

28. Hu, K.; Jin, G.-J.; Mei, W.-C.; Li, T.; Tao, Y.-S. Increase of medium-chain fatty acid ethyl ester content in mixed H. uvarum/S. cerevisiae fermentation leads to wine fruity aroma enhancement. Food Chem. 2018, 239, 495–501. [CrossRef]

(13)

29. Nevado, F.P.; Albergaria, H.; Hogg, T.; Gírio, F. Cellular death of two non-Saccharomyces wine-related yeasts during mixed fermentations with Saccharomyces cerevisiae. Int. J. Food Microbiol. 2006, 108, 336–345. 30. Lage, P.; Barbosa, C.; Mateus, B.; Vasconcelos, I.; Mendes-Faia, A.; Mendes-Ferreira, A. H. guilliermondii

impacts growth kinetics and metabolic activity of S. cerevisiae: The role of initial nitrogen concentration. Int. J. Food Microbiol. 2014, 172, 62–69. [CrossRef]

31. Viana, F.; Belloch, C.; Vallés, S.; Manzanares, P. International Journal of Food Microbiology Monitoring a mixed starter of Hanseniaspora vineae—Saccharomyces cerevisiae in natural must: Impact on 2-phenylethyl acetate production. Int. J. Food Microbiol. 2011, 151, 235–240. [CrossRef]

32. Medina, K.; Boido, E.; Dellacassa, E.; Carrau, F. Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomyces cerevisiae during wine fermentation. Int. J. Food Microbiol. 2012, 157, 245–250. [CrossRef] 33. Medina, K.; Boido, E.; Farina, L.; Gioia, O.; Gómez, M.; Barquet, M.; Gaggero, C.; Dellacassa, E.; Carrau, F.

Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae. Food Chem. 2013, 141, 2513–2521. [CrossRef] [PubMed]

34. Lleixà, J.; Martín, V.; Portillo, M.D.C.; Carrau, F.; Beltran, G.; Mas, A. Comparison of Fermentation and Wines Produced by Inoculation of Hanseniaspora vineae and Saccharomyces cerevisiae. Front. Microbiol. 2016, 7, 338. [CrossRef] [PubMed]

35. Cocolin, L.; Bisson, L.; Mills, D. Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiol. Lett. 2000, 189, 81–87. [CrossRef] [PubMed]

36. Contreras, A.; Hidalgo, C.; Schmidt, S.; Henschke, P.; Curtin, C.; Varela, C. The application of non-Saccharomyces yeast in fermentations with limited aeration as a strategy for the production of wine with reduced alcohol content. Int. J. Food Microbiol. 2015, 205, 7–15. [CrossRef]

37. Ruiz, J.; Belda, I.; Beisert, B.; Navascués, E.; Marquina, D.; Calderón, F.; Benito, S. Analytical impact of Metschnikowia pulcherrima in the volatile profile of Verdejo white wines. Appl. Microbiol. Biotechnol. 2018, 102, 8501–8509. [CrossRef]

38. Bely, M.; Stoeckle, P.; Masneuf-Pomarede, I.; Dubourdieu, D. Impact of mixed Torulaspora delbrueckii– Saccharomyces cerevisiae culture on high-sugar fermentation. Int. J. Food Microbiol. 2008, 122, 312–320. [CrossRef]

39. Renault, P.; Coulon, J.; De Revel, G.; Barbe, J.-C.; Bely, M. Increase of fruity aroma during mixed T. delbrueckii/S. cerevisiae wine fermentation is linked to specific esters enhancement. Int. J. Food Microbiol. 2015, 207, 40–48. [CrossRef]

40. Azzolini, M.; Fedrizzi, B.; Tosi, E.; Finato, F.; Vagnoli, P.; Scrinzi, C.; Zapparoli, G. Effects of Torulaspora delbrueckii and Saccharomyces cerevisiae mixed cultures on fermentation and aroma of Amarone wine. Eur. Food Res. Technol. 2012, 235, 303–313. [CrossRef]

41. Taillandier, P.; Lai, Q.P.; Julien-Ortiz, A.; Brandam, C. Interactions between Torulaspora delbrueckii and Saccharomyces cerevisiae in wine fermentation: Influence of inoculation and nitrogen content. World J. Microbiol. Biotechnol. 2014, 30, 1959–1967. [CrossRef]

42. Tondini, F.; Lang, T.; Chen, L.; Herderich, M.; Jiranek, V. Linking gene expression and oenological traits: Comparison between Torulaspora delbrueckii and Saccharomyces cerevisiae strains. Int. J. Food Microbiol. 2019, 294, 42–49. [CrossRef]

43. Canonico, L.; Comitini, F.; Ciani, M. Influence of vintage and selected starter on Torulaspora delbrueckii/Saccharomyces cerevisiae sequential fermentation. Eur. Food Res. Technol. 2015, 241, 827–833. [CrossRef]

44. Mora, J.; Barbas, J.I.; Mulet, A. Growth of Yeast Species during the Fermentation of Musts Inoculated with Kluyveromyces thermotolerans and Saccharomyces cerevisiae. Am. J. Enol. Vitic. 1990, 41, 156–159.

45. Kapsopoulou, K.; Mourtzini, A.; Anthoulas, M.; Nerantzis, E. Biological acidification during grape must fermentation using mixed cultures of Kluyveromyces thermotolerans and Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 2007, 23, 735–739. [CrossRef]

46. Gobbi, M.; Comitini, F.; Domizio, P.; Romani, C.; Lencioni, L.; Mannazzu, I.; Ciani, M. Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine. Food Microbiol. 2013, 33, 271–281. [CrossRef] [PubMed]

47. Benito, Á.; Calderón, F.; Palomero, F.; Benito, S. Quality and Composition of Airén Wines Fermented by Sequential Inoculation of Lachancea thermotolerans and Saccharomyces cerevisiae. Food Technol. Biotechnol. 2016, 54, 135–144. [CrossRef]